Reduced Enzyme Dynamics upon Multipoint Covalent Immobilization Leads to Stability-Activity Trade-off

Abstract

The successful incorporation of enzymes into materials through multipoint covalent immobilization (MPCI) has served as the foundation for numerous advances in diverse fields, including biocatalysis, biosensing, and chemical weapons defense. Despite this success, a mechanistic understanding of the impact of this approach on enzyme stability has remained elusive, which is critical for realizing the full potential of MPCI. Here, we showed that the stabilization of lipase upon MPCI to polymer brush surfaces resulted from the rigidification of the enzyme with an increase in the number of enzyme-brush attachments. This was evident by a 10-fold decrease in the rates of enzyme unfolding and refolding as well as a reduction of the intrinsic fluctuations of the folded and unfolded states, which was measured by single-molecule (SM) Förster Resonance Energy Transfer imaging. Moreover, our results illuminate an important trade-off between stability and activity as a function of this decrease in structural dynamics of the immobilized lipase. Notably, as the thermal stability of lipase increased, as indicated by the temperature optimum for activity of the enzyme, the specific activity of lipase decreased. This decrease in activity was attributed to a reduction in the essential motions of the folded state that are required for catalytic turnover of substrate. These results provide direct evidence of this effect, which has long been a matter of speculation. Furthermore, our findings suggest that the retention of activity and stabilization of an enzyme may be balanced by tuning the extent of enzyme attachment.

Mapping protein–polymer conformations in bioconjugates with atomic precision

“… 12 In a subsequent publication, by the same group, it was found that there is a trade-off in this system – more protein–polymer attachments resulted in higher stability but lower activity due to decreased motion in the folded state of the protein. 13 This experimental result conflicts with recent work on soluble chymotrypsin conjugates, where MD models showed more motion in the conjugate with the lowest enzymatic activity, a manifestation of a measured decrease in the substrate binding constant, K m . 14 …”

Implicit modeling of the impact of adsorption on solid surfaces for protein mechanics and activity with a coarse-grain representation

“…In particular, our results highlight the fact that the structural integrity of surface immobilized enzymes is no guarantee that the enzyme will remain functional, and that the systems' internal dynamics should be specifically investigated before drawing any conclusions regarding the conservation of the catalytic activity. The mechanical perturbations obtained for RNase A grafted on various surfaces (and with various orientations) illustrate the stability-activity tradeoff that was recently observed13 by Weltz et al(53) using single-molecule FRET imaging. Altogether our modeling approach provides a simple tool to help understand how protein adsorption might impact their dynamics and function, especially when one needs to achieve a delicate balance between an enzyme stability and its catalytic activity.…”

Characterization and evaluation of immobilized enzymes for applications in flow reactors

“…The enzyme stability relies both on the reversibility and valency of the enzyme-carrier interaction. It is widely accepted [6,7] and lastly supported by singlemolecule experiments [53], that irreversible multivalent attachments contribute to the structural stabilization of immobilized enzymes. Semproli et al demonstrated that one transaminase from Vibrio fluvialis immobilized through glyoxyl chemistry on agarose porous microbeads works 22 fold more efficiently in flow (5.35 mg product x mg enzyme -1 x h -1 ) than in batch [54].…”